The authors would like to thank the National Science Foundation for the continuous financial support (CHE-0742365) and for the purchase of a Bruker APEX II diffractometer (CHE-0443233) and a Bruker Microtof-II mass spectrometer (CHE-0741793). The authors would also like to thank CEST facility for their use of the Micromass Quattro-LC mass spectrometer.

1. Preparing Niobium Tubes
<ol>
 <li>Before cutting the niobium (Nb) tubes, prepare Nb-cleaning solution. In a 500 ml plastic bottle, measure out and add via a 100 ml measuring cylinder the following stock solutions as received: 110 ml H2SO4, followed by 50 ml HNO3, followed by 40 ml HF. Mix well and allow reaching room temperature before using.</li>
 <li>Measure the Nb tube, 4.5 cm in length, and cut with a pipe cutter. Avoid kinking the tubing. Repeat 3 more times.</li>
 <li>In a well-ventilated fume-hood, place the four Nb tubes, length-wise, in a 100 ml plastic beaker. Pour the Nb-cleaning solution into the beaker until tubes are completely covered. Close the sash of the fume-hood. Wait 12 to 20 seconds, or until brown nitrous oxide gas is released. Immediately fill the beaker with water and allow reaching room temperature.</li>
 <li>Use plastic thongs to remove Nb tubes, wash several times with water and then acetone, and dry them in a high temperature drying oven. (Dispose of the used acid solution after carefully neutralizing with KOH).</li>
 <li>Take the Nb tubes out of the oven and allow cooling down to room temperature. With vise-grips crimp about 1 cm of one end of each tube. Bend slightly the edge, about 1 mm.</li>
 <li>Turn on the vacuum pump and argon gas cylinder of the arc-welder. Clamp the four Nb tubes, in a staggered position, in the arc-welder's holder. Center the brass block (heat sink) in the arc-welder and slowly adjust niobium tubes on top. Close and clamp shut the door of arc welder.</li>
 <li>Adjust welding tip to height of niobium tubes. Turn on vacuum valve (ensure that argon refill valve is close). Allow vacuum to reach below 100 mm Hg (ideal: 30 mm Hg; wait time: 30 - 40 minutes) in order to completely evacuate chamber and Nb tubes of all air and moisture.</li>
 <li>When 30 - 60 mm Hg vacuum is achieved, close vacuum valve and slowly open the valve to argon cylinder to refill with argon to 7 in. Hg. Close argon gas valve. System should now be under partial vacuum.</li>
 <li>Put on certified rubber welding gloves. Position protective welding darkened glass over the window of the arc welder. Turn on the source set at 15 A (thicker niobium tubes use 20 - 55 A). As welding tip makes contact with niobium tube, welding arc forms, raise 1 cm over tube and slowly sweep above tube as niobium melts and seals completely. Repeat for remaining niobium tubes. Turn off power source and allow system to reach room temperature (15 - 30 min).</li>
 <li>Remove clamp from arc welder door. Slowly open argon gas valve and refill with argon until door is opened.</li>
 <li>Remove niobium tubes and label with electrical engraver: A, B, C, and D, respectively. Place back in drying oven until dry and place hot in antechamber of drybox, pumping under vacuum for 30 - 45 min.</li>
 <li>Refill small antechamber with high-purity nitrogen/argon gas. Bring four labeled niobium tubes into drybox. The containers are now ready to be loaded.</li>
</ol>
2. Loading Niobium Tubes: Preparing K4Ge9
<ol>
 <li>Tare the balance and then measure 156 mg K (4 mmol). With a spatula, carefully insert it into the Nb tube and push to the bottom.</li>
 <li>Weigh 653 mg of Ge (9 mmol) and insert into the tube on top of potassium metal.</li>
 <li>Carefully crimp the open end of the Nb tube using vise-grips and slightly bend the edge. Repeat for the three other Nb tubes. Place all four tubes in a jar [under inert gas], close the jar and place it in the box antechamber in order to bring out.</li>
 <li>Following steps 1.5 - 1.8, weld edges of four Nb tubes. The loaded tubes are now ready to be sealed in a fused silica tube.</li>
</ol>
3. Preparing Fused Silica Tube via Glass-Blowing
<ol>
 <li>Use circular glass saw to cut 10-14" pieces of large quartz tube (ID/OD = 20/22 mm) and medium quartz tube (7/9 mm). Cut also end of round ball joint. Wash thoroughly with water and acetone. Place in glass drying oven until completely dry. Take out of oven and allow reaching room temperature.</li>
 <li>Light the hydrogen/oxygen torch with a striker and slowly increase oxygen flow to get hot blue flame. Put on darkened safety glasses before inserting any quartz tubing into flame.</li>
 <li>Insert the large tube in the flame and slowly rotate one end allowing the tube to collapse to a bottle neck and then completely close. Using a spare medium quartz tube as a shaping rod, pull one end of the "white hot" (as seen through safety glasses) quartz tube to close the opening more quickly.</li>
 <li>After sealing opening, attach rubber septum with a blow hose at the open end and insert blow pipe into mouth (do not blow yet). While rotating the sealed end in the flame, slightly blow to keep positive pressure and prevent glass from collapsing. Next, a small hole is ready to be made in the center.</li>
 <li>Adjust gas flow to create a sharp blue flame and focus flame in center of closed end. When targeted spot becomes white hot, blow hard to create a large bubble or opening. Break bubble by gently scraping on lab bench. Insert opened hole into flame and open to 0.7 - 1cm while flaring edges with graphite reamer. This part (body) is now ready to be attached to the smaller tubing (neck).</li>
 <li>Close one end of the neck piece with rubber septum. Holding in one hand the body (with mouthpiece in mouth, be ready to blow) and in other hand the neck slowly rotate both ends simultaneously in the flame. Attach the ends and straighten outside of flame.</li>
 <li>Adjust size of flame to sharp one and slowly work out each quarter of sealed joint to ensure no holes and air bubbles. Keep positive pressure in order to prevent glass from collapsing. Remove the septum from top of neck. The neck is now ready to be attached to the ball joint.</li>
 <li>Using septum, close ball joint opening. Attach end of ball joint to neck following steps 3.5 - 3.6.</li>
 <li>Insert four Nb tubes into opened end of body. Attach end of blow hose to ball joint. Slowly close opened end of body by following step 3.3. Turn off flame and close all gas cylinders. Allow fused silica/quartz jacket to cool down.</li>
 <li>Pour dilute Nb cleaning solution (2 H20: 1 solution, refer to 1.1) into the silica tube and keep for 3 - 5 minutes until the Nb tubes are shiny and free of any oxidized areas. Rinse three times each with distilled water and acetone. Allow to dry completely. The niobium tubes are now in a fused silica/quartz jacket and ready to be evacuated and sealed off.</li>
</ol>
4. Sealing Fused Silica Tube using a High Vacuum Line
<ol>
 <li>Turn on the pump for the vacuum line. Fill dewars with liquid nitrogen. Turn on the mercury diffusion pump and the cooling water. Allow to reflux for a minimum of 30 minutes.</li>
 <li>Coat ball joint of fused silica jacket with high vacuum silicone grease. Attach to vacuum line. Evacuate tube completely for 30 min.</li>
 <li>Using a tesla coil, check for any leaks/holes.</li>
 <li>Put on Borosilicate Safety Glasses. Turn on small house gas/oxygen torch. Slowly pass flame from bottom of tube to neck, ensuring all air and moisture is gone.</li>
 <li>Slowly pass flame over Nb tubes, heating gently for 1 - 2 minutes. Careful not to get glass white hot as it will collapse as it is pulled in by high vacuum. Turn off torch and allow fused silica jacket to reach room temperature (15 - 30 min).</li>
 <li>Repeat step 4.4. Intensify flame to a sharp hot blue color. Slowly seal off tube below ball and neck joint. (Tip: can get entire glass area white hot and assist by slowing pulling off with other hand by holding end of silica jacket). Turn off torch.</li>
 <li>Turn off Hg-distillation. Allow to reach room temperature. Turn off water. Allow liquid nitrogen to slowly evaporate. The fused silica jacket is now ready to be placed in the furnace.</li>
</ol>
5. Heating the Reaction Mixtures in a Furnace
<ol>
 <li>Gently bring niobium tubes to center of quartz jacket and place directly over thermocouple. Insulate openings of furnace with glass wool. Turn on furnace and set at 950 &deg;C for two days.</li>
 <li>After completion, using the glass-cutting circular saw (refer to 3.1) cut open the end of the fused silica tube and remove the Nb containers. Wash the niobium free of any debris with water and acetone and dry in the oven. Pump into the drybox.</li>
 <li>Using wire-cutting pliers cut the ends of the niobium tubes. Gently crush the coarse product. The fine intermetallic precursor is now ready to be brought into solution.</li>
</ol>
6. Dissolving Precursor in Ethylenediamine
<ol>
 <li>Weigh out 81 mg (0.1 mmol) of K4Ge9 into a test-tube. Add stir bar.</li>
 <li>Pipette in 2 - 2.5 ml of anhydrous ethylenediamine (previously distilled over sodium metal) and stir for 5 - 10 min at room temperature. A bright red solution forms.</li>
 <li>Note: If solution is stirred for too long, it will become green, indicative of oxidized clusters. A red solution is desired for functionalization.</li>
</ol>
7. Reacting Ge9-clusters with Me3SiC&equiv;CSiMe3
<ol>
 <li>Slowly syringe in, drop-wise, 0.056 ml (0.25 mmol; a little over 2 equivalents) of Me3SiC&equiv;CSiMe3. Oily layer is seen on top of the red cluster solution. As the reaction is stirred, the solution slowly becomes brown. Stir for four hours until a clear honey-brown solution is achieved.</li>
 <li>Place test-tube in centrifuge for 15 min. Carefully remove. Insert a glass fiber filter pipette (previously packed, dried at 180 &deg;C overnight and pumped into drybox) into a clean test-tube.</li>
 <li>Carefully pipette the supernatant solution into the filter pipette and allow to filter. The clear honey-brown filtrate is now collected.</li>
 <li>Set aside 0.1 ml of filtrate in order to run an electrospray mass spectrometry (ES-MS) sample to confirm product before crystallization.</li>
</ol>
8. Running ES-MS of Reaction Solution
<ol>
 <li>Pump a clean 1 ml Hamilton syringe, syringe plunger and PEEK tubing into antechamber of drybox, for at least 30 - 45 min.</li>
 <li>Insert syringe plunger into syringe and fill with anhydrous ethylenediamine and dispense into a waste container. Purge the syringe two more times.</li>
 <li>Fill the syringe again with anhydrous ethylenediamine and push through the PEEK tubing. Purge the PEEK tubing two more times. The PEEK tubing is now filled with anhydrous ethylenediamine.</li>
 <li>Fill the empty syringe with the filtered Ge9-divinyl solution. (Tip: this can be placed in a nitrogen filled Ziploc bag for transportation if mass spectrometer is outside of the drybox). Bring out of drybox.</li>
 <li>Place syringe attached to PEEK tubing in Harvard syringe pump at 10 &mu;L/min. Attach PEEK tubing securely to electrospray mass spectrometer. Collect spectrum in the negative ion mode on a Micromass Quattro-LC triple quadrupole mass spectrometer (typical conditions: 100 &deg;C source temperature, 125 &deg;C desolvation temperature, 2.5 kV capillary voltage, 30 - 65 V cone voltage) or on a Bruker Microtof-II mass spectrometer (capillary at 3800 V, nebulizer at 0.6 Bar, desolvation temperature at 190 &deg;C, capillary exit at 100 V, detector at 1200 V). (Note: samples are highly air and moisture sensitive, purging the chamber for 15 - 60 min before running samples is preferred.)</li>
 <li>Resulting spectrum will show isotope pattern for the functionalized clusters (see Figure 1). The remaining filtrate in the drybox can now be crystallized.</li>
</ol>
9. Crystallizing Ge9-divinyl Ions with a Sequestering Agent
<ol>
 <li>Pipette remaining filtrate into two equal aliquots in two clean test-tubes. Label test-tubes C and D (as an example). Set aside.</li>
 <li>Weigh 0.4 mmol (105 mg) of anhydrous 18-crown-6 into a clean test-tube and add 8 ml of toluene. Mix well until completely dissolved.</li>
 <li>Layering with Toluene, Method A: Gently pipette 4 ml of this solution on top of the test-tube C and D. (Tip: Try to achieve two separate phases.) Place a rubber stopper on each test-tube. Set aside undisturbed in a test-tube rack to crystallize.</li>
 <li>Layering with Toluene, Method B: Pipette 4ml of 18-crown-6/toluene solution into two clean test-tube labeled E and D. Insert a filter pipette (note: if solution needs to be filtered again) or a regular pipette into test-tube E and F. Pipette the solution of test-tube C into E and test-tube D into F. (Note: this reverse layering will quickly result in two phases.) Place a rubber stopper on each test-tube. Set aside undisturbed in a test-tube rack to crystallize.</li>
 <li>Bright orange blocks should crystallize within 1 - 3 days. The unit cell of the crystals can then be confirmed by single crystal X-ray diffraction.</li>
</ol>
10. Checking Crystals Unit Cell on a D8-Diffractometer
<ol>
 <li>Fill a plastic bottle with Paratone-N oil and allow all air bubbles to be removed. Completely degas the oil in antechamber of drybox, under vacuum, overnight. Bring into the drybox.</li>
 <li>Apply 2 - 3 drops to glass slide. Coat spatula tip in the oil and insert into crystallizing test-tube. Select orange crystals from the test-tube and immersed in the Paratone-N oil. (Note: ensure all air-sensitive crystals are coated in oil). Bring outside drybox.</li>
 <li>Using a high resolution microscope, select a single crystal and drag to the edge of the oil on the slide with a straight stainless probe.</li>
 <li>Carefully remove excess oil from the crystal by dragging to edge of glass slide. Mount single crystal on Mitegen micro mount loop and quickly position under cold stream (100 K) of the Bruker D8 APEX-II diffractometer equipped with CCD area detector using graphite-monochromated Mo K&alpha; radiation.</li>
 <li>Ensure good high angle diffractions and acquire unit cell.</li>
 <li>Compare and confirm unit cell to that of [K(18-crown-6)]2[Ge9(HCCH2)2]&bull;en, 1, triclinic, P-1, a = 10.974(4), b = 14.3863(5), and c = 16.2272(6) &Aring;, &alpha; = 85.946(2), &beta; = 71.136(2), and &gamma; = 89.264(2) &deg;, V = 2412.21(15) &Aring;3, Z =2. 53</li>
</ol>
11. Representative Results
The unique isotope pattern of the anionic clusters allows them to be easily detected in the negative ion-mode (Fig 1). Also noteworthy is that reduced singly charged species, in addition to pairing with a potassium ion is a common phenomenon of this soft-ionization technique.59
The crystal structure with relevant bond lengths and angles of [Ge9(CH=CH2)2]2- in [K(18-crown-6)]2[Ge9(HCCH2)2]&bull;en, 1, can be seen in Figure 2 .
<img alt="Figure 1" src="/files/ftp_upload/3532/3532fig1.jpg" /> 
Figure 1. ES-MS spectra (negative-ion mode) of ethylenediamine solutions of the reactions of Ge9 clusters with Me3SiC&equiv;CSiMe3. Shown are also the theoretical isotope distributions below the experimental distribution. (Sevov et. al. Inorg. Chem. 2007, 46, 10953.)
<img alt="Figure 2" src="/files/ftp_upload/3532/3532fig2.jpg" /> 
Figure 2. A view of [K(18-crown-6)]2[Ge9(HCCH2)2]&bull;en, 1. Color scheme: <img alt="Bullet 1" src="/files/ftp_upload/3532/3532bullet1.jpg" /> = Ge, <img alt="Bullet 2" src="/files/ftp_upload/3532/3532bullet2.jpg" /> = C, <img alt="Bullet 3" src="/files/ftp_upload/3532/3532bullet3.jpg" /> = H. Selected bond lengths and angles: Ge-C 1.961 and 1.950 &Aring;, C=C 1.318 and 1.316 &Aring;, Ge-C-C 123 and 127 &deg;. (Sevov et. al. Inorg. Chem. 2007, 46, 10953.)
<img alt="Figure 3" src="/files/ftp_upload/3532/3532fig3.jpg" /> 
Figure 3. Schematic representation of Preparing Niobium Tubes: (a) cutting Nb tubes; (b) cleaning Nb tubes in a Nb acid solution; (c) using vise-grips to crimp and bend Nb tube.
<img alt="Figure 4" src="/files/ftp_upload/3532/3532fig4.jpg" /> 
Figure 4. Schematic representation of Preparing Niobium Tubes: (a) diagram of arc welder; (b) staggered Nb tubes in arc welder holder and (c) welding tip above Nb tubes.
<img alt="Figure 5" src="/files/ftp_upload/3532/3532fig5.jpg" /> 
Figure 5. Schematic representation of Loading Niobium Tubes : (a) inside the drybox and (b) Nb tubes: (i) before welding, (ii) after using vise-grips to crimp one edge, (iii) after welding one edge, (iv) after loading and then welding the Nb tube closed, (v) after opening the Nb tube to take out the K4Ge9 precursor.
<img alt="Figure 6" src="/files/ftp_upload/3532/3532fig6.jpg" /> 
Figure 6. Schematic representation of Preparing Fused-Silica Tube by Glass-Blowing in (a) and (b)(i) large and small quartz tubing, (ii) body and neck sealed together, (iii) neck with ball joint, (iv) neck and ball joint sealed together, (v) Nb tubes sealed inside quartz tube, (vi) after the fused silica tube is sealed.
<img alt="Figure 7" src="/files/ftp_upload/3532/3532fig7.jpg" /> 
Figure 7. Schematic representation of Sealing Fused Silica Tubes on a High Vacuum Line in (a) and (b) after the Nb tubes is sealed showing etching of the quartz tube from Nb acid solution.
<img alt="Figure 8" src="/files/ftp_upload/3532/3532fig8.jpg" /> 
Figure 8. Schematic representation of Placing Loaded Fused Silica Tubes in Furnace.
<img alt="Figure 9" src="/files/ftp_upload/3532/3532fig9.jpg" /> 
Figure 9. Schematic representation of Reacting K4Ge9 with Me3SiC&equiv;CSiMe3 inside the drybox (a) (i) unopened Nb tube, (ii) one edge of the Nb tube cut with the (iii) cutting pliers, (iv) crushed precursor and (b) (i) precursor dissolved in ethylenediamine, (ii) immediately after Me3SiC&equiv;CSiMe3 is added (oily droplets on top of test-tube walls seen).
<img alt="Figure 10" src="/files/ftp_upload/3532/3532fig10.jpg" /> 
Figure 10. Schematic representation of Running ES-MS of Reaction Solution in (a) mass spectrometer syringe prepared in dry box, (b) Bruker Microtof-II.
<img alt="Figure 11" src="/files/ftp_upload/3532/3532fig11.jpg" /> 
Figure 11. Schematic representation of Crystallizing Ge9-divinyl with Sequestering Agents in (a) reverse layering and (b) several hours later.
<img alt="Figure 12" src="/files/ftp_upload/3532/3532fig12.jpg" /> 
Figure 12. Schematic representation of Checking Crystals Unit Cell on a D8-Diffractometer: (a) selecting crystals under the microscope and (b) collecting a unit cell.

No conflicts of interest declared.

It is important to clean well the partially oxidized Nb tubes. However, if the tubes are left too long in the Nb cleaning solution, this will severely compromise the thickness of the tube. Thus, 10 &#x2013; 15 seconds are imperative and the tubes should be very lustrous at the end (Fig 3). After the tubes are sealed inside the fused silica jacket they should be cleaned again with a dilute Nb acid solution. This should result in mild effervescence, cleaning any oxidized areas on Nb tubes that occurred during welding o...

<table border="1">
<tbody>
<tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>D8-Xray diffractometer</td>
 <td>Bruker</td>
 <td>Bruker APEX II</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Electrospray mass spectrometer</td>
 <td>Bruker</td>
 <td>Microtof-II</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Electrospray mass spectrometer</td>
 <td>Micromass</td>
 <td>Quattro-LC</td>
 <td>triple -quadropole</td>
 </tr>
 <tr>
 <td>Drybox</td>
 <td>Innovative Technology</td>
 <td>S-1-M-DL</td>
 <td>IT-Sys1 model</td>
 </tr>
 <tr>
 <td>Inert Gas/Vacuum Shielded 
 Arc Welding Arrangement</td>
 <td><a href="http://www.ldsvacuum.com" target="_blank">LDS Vacuum Products</a> </td>
 <td>Special Order</td>
 <td></td>
 </tr>
 <tr>
 <td>Arc Welder Power Source</td>
 <td>Miller</td>
 <td>Maxstar-91</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Welding Rubber Gloves</td>
 <td>The Home Depot</td>
 <td>&nbsp;KH643</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Electric Engraver</td>
 <td>Burgess Products</td>
 <td>74</td>
 <td>Vibro-Graver</td>
 </tr>
 <tr>
 <td>Circular Glass Saw</td>
 <td>Pistorius Machine Co. Inc</td>
 <td>GC-12-B</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Tube Furnace</td>
 <td>Lindberg/Blue M</td>
 <td>TF55035</td>
 <td>Minimite Laboratory Tube Furnace, Moldatherm (1100 &deg;C)</td>
 </tr>
 <tr>
 <td>Glass Drying Oven</td>
 <td>Fisher Scientific</td>
 <td>13-247-650G</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>High Vacuum Hg Schlenk-Line</td>
 <td> Special Order</td>
 <td>Univ Of Notre Dame</td>
 <td>Alternative: Edwards E050/60; VWR International; Cat. No. EVB302-07-110</td>
 </tr>
 <tr>
 <td>Large Torch</td>
 <td>Victor</td>
 <td>JT100C</td>
 <td>Welding torch, tip: Victor 5-W-J </td>
 </tr>
 <tr>
 <td>Small Torch</td>
 <td>Veriflo Co.</td>
 <td>3A</td>
 <td>Blow-pipe </td>
 </tr>
 <tr>
 <td>Tesla Coil</td>
 <td>VWR International</td>
 <td>KT691550-0000</td>
 <td>Leak detector</td>
 </tr>
 <tr>
 <td>Stirrer/Hot -Plate</td>
 <td>VWR International</td>
 <td>12620-970</td>
 <td>VWR HOT PLATE STR DY-DUAL120V</td>
 </tr>
 <tr>
 <td>Balance</td>
 <td>Denver Instrument Co.</td>
 <td>100A</td>
 <td>XE Series</td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>LW Scientific</td>
 <td>E8C-08AV-1501</td>
 <td>Variable speed</td>
 </tr>
 <tr>
 <td>Graphite Reamer, (flaring)</td>
 <td>ABR Imagery, Inc.</td>
 <td>850-523 B01</td>
 <td>Open holes in Glass Blowing and flaring edges</td>
 </tr>
 <tr>
 <td>Striker</td>
 <td>Fisher Scientific</td>
 <td>12-007</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Vise-Grips</td>
 <td>The Home Depot</td>
 <td>0902L3SM</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Pipe-Cutter</td>
 <td>The Home Depot</td>
 <td>32820</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Cutting Pliers</td>
 <td>The Home Depot</td>
 <td>437</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Plastic Beaker</td>
 <td>VWR International</td>
 <td>13890-046</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Measuring Cylinder</td>
 <td>VWR International</td>
 <td>65000-006</td>
 <td>Careful, HF etches glass (if using a glass one)</td>
 </tr>
 <tr>
 <td>Large Plastic Bottle</td>
 <td>VWR International</td>
 <td>16128-542</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>13 x 100 Test-Tubes</td>
 <td>VWR International</td>
 <td>47729-572</td>
 <td>CULTURE TUBE 13X100 CS1000</td>
 </tr>
 <tr>
 <td>Laboratory (Rubber) Stoppers</td>
 <td>Sigma Chemical Co.</td>
 <td>Z164437-100EA</td>
 <td>Size 00</td>
 </tr>
 <tr>
 <td>Test-Tube Rack</td>
 <td>VWR International</td>
 <td>60196-702</td>
 <td>10-13 mm tube OD</td>
 </tr>
 <tr>
 <td>Stir-Bars</td>
 <td>StirBars.com/Big Science Inc.</td>
 <td>SBM-0803-MIC</td>
 <td>PTFE 8x3 mm Micro</td>
 </tr>
 <tr>
 <td>Glass Pipettes</td>
 <td>VWR International</td>
 <td>14673-043</td>
 <td>VWR PIPET PASTEUR 9IN CS1000</td>
 </tr>
 <tr>
 <td>Rubber Bulbs</td>
 <td>VWR International</td>
 <td>56311-062</td>
 <td>Latex, thin walled</td>
 </tr>
 <tr>
 <td>Glass Wool</td>
 <td>Unifrax I LLC</td>
 <td>6048</td>
 <td>Fiberfrax Bulk Fiber Insulation, Ceramic fiber</td>
 </tr>
 <tr>
 <td>Glass Slides</td>
 <td>VWR International</td>
 <td>16004-422</td>
 <td>75x25x1mm, Microscope Slides</td>
 </tr>
 <tr>
 <td>Paratone-N oil</td>
 <td>Hampton Research</td>
 <td>Parabar 10312</td>
 <td>Known as: Paratone-N, Paratone-8277, Infineum V8512</td>
 </tr>
 <tr>
 <td>High Vacuum Silicone Grease</td>
 <td>VWR International</td>
 <td>59344-055</td>
 <td>Dow Corning</td>
 </tr>
 <tr>
 <td>Liquid Nitrogen</td>
 <td>Univ. of Notre Dame</td>
 <td>&nbsp; </td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Argon Gas Cylinder</td>
 <td>Praxair Distribution Inc.</td>
 <td>TARGHP </td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Nitrogen Gas Cylinder</td>
 <td>Praxair Distribution Inc.</td>
 <td>QNITPP</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Oxygen Gas Cylinder</td>
 <td>Praxair Distribution Inc.</td>
 <td>OT</td>
 <td>337 cf CYL</td>
 </tr>
 <tr>
 <td>Hydrogen Gas Cylinder</td>
 <td>Praxair Distribution Inc.</td>
 <td>HK</td>
 <td>195 cf CYL</td>
 </tr>
 <tr>
 <td>Propane Gas Cylinder/source</td>
 <td>Univ. of Notre Dame</td>
 <td>UND</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Quartz tubing, Lg</td>
 <td>Quartz Scientific Inc.</td>
 <td>100020B</td>
 <td>20 mm id x 22mm od x 48" clear fused quart tubing</td>
 </tr>
 <tr>
 <td>Quartz tubing, Md</td>
 <td>Quartz Scientific Inc.</td>
 <td>100007B</td>
 <td>Clear Fused Quartz Tubing,7mm id x 9mm od x 48"</td>
 </tr>
 <tr>
 <td>Round Bottom Quartz Joint</td>
 <td>Quartz Scientific Inc.</td>
 <td>6160189B</td>
 <td>Ball joint</td>
 </tr>
 <tr>
 <td>Quartz Safety Glasses</td>
 <td>Wale Apparatus</td>
 <td>11-1127</td>
 <td>waleapparatus.com</td>
 </tr>
 <tr>
 <td>Pyrex Safety Glasses</td>
 <td>Wale Apparatus</td>
 <td>11-2125-B3</td>
 <td>For clear and color borosilicate glass</td>
 </tr>
 <tr>
 <td>Blow Hose Kit</td>
 <td>Glass House</td>
 <td>BH020</td>
 <td>glasshousesupply.com</td>
 </tr>
 <tr>
 <td>Niobium Tubes</td>
 <td>Shaanxi Tony Metals Co., Ltd</td>
 <td>Niobium Tube, 50 ft</td>
 <td>Seamless Niobium Tube Outside diameter: 0.375 (&plusmn;0.005) inches. 
 Wall thickness: 0.02(&plusmn;0.003) Inches Niobium should be annealed.</td>
 </tr>
 <tr>
 <td>PEEK Starter Kit for Mass Spect</td>
 <td>Waters</td>
 <td>PSL613321</td>
 <td>PEEK (PolyEtherEtherKetone) tubing, nuts, ferrule, fits</td>
 </tr>
 <tr>
 <td>Mass Spect Needle Set</td>
 <td>VWR International</td>
 <td>60373-992</td>
 <td>Hamilton Manufacturer (81165)</td>
 </tr>
 <tr>
 <td>H2SO4</td>
 <td>VWR International</td>
 <td>BDH3072-2.5LG</td>
 <td>ACS Grade</td>
 </tr>
 <tr>
 <td>HNO3</td>
 <td>VWR International</td>
 <td>BDH3046-2.5LPC</td>
 <td>ACS Grade</td>
 </tr>
 <tr>
 <td>HF</td>
 <td>VWR International</td>
 <td>BDH3040-500MLP</td>
 <td>ACS Grade</td>
 </tr>
 <tr>
 <td>Distilled Water</td>
 <td>Univ. of Notre Dame</td>
 <td>UND</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Acetone</td>
 <td>VWR International</td>
 <td>BDH1101-4LP</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Ethylenediamine</td>
 <td>VWR International</td>
 <td>AAA12132-0F</td>
 <td>99% 2.5 L</td>
 </tr>
 <tr>
 <td>Toluene</td>
 <td>VWR International</td>
 <td>200004-418</td>
 <td>99.8 %, anhydrous</td>
 </tr>
 <tr>
 <td>Mercury</td>
 <td>Strem Chemicals, Inc.</td>
 <td>93-8046</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Potassium (K) metal</td>
 <td>Strem Chemicals, Inc.</td>
 <td>19-1989</td>
 <td>Sealed in glass ampoule under Ar</td>
 </tr>
 <tr>
 <td>Germanium (Ge) powder</td>
 <td>VWR International</td>
 <td>AA10190-18</td>
 <td>GERM PWR -100 MESH 99.999% 50G</td>
 </tr>
 <tr>
 <td>Bistrimetylsilylacetylene, 
 (Me3SiC&equiv;CCSiMe3)</td>
 <td>Fischer Scientific</td>
 <td>AC182010100</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>18-crown-6 
 (1,4,7,10,13,16-Hexaoxacyclooctadecane)</td>
 <td>VWR International</td>
 <td>200001-954</td>
 <td>99%, 25 gm</td>
 </tr>
 <tr>
 <td>2,2,2-crypt 
 (4,7,13,16,21,24-Hexaoxa-1,10 diazabicyclo[8.8.8]hexacosane)</td>
 <td>Sigma Aldrich </td>
 <td>291110-1G</td>
 <td>98%</td>
 </tr>
 </tbody>
</table>

synthesis of nine-atom deltahedral zintl ions of germanium and their functionalization with organic groups

Although the first studies of Zintl ions date between the late 1890's and early 1930's they were not structurally characterized until many years later.1,2 Their redox chemistry is even younger, just about ten years old, but despite this short history these deltahedral clusters ions E9n- (E = Si, Ge, Sn, Pb; n = 2, 3, 4) have already shown interesting and diverse reactivity and have been at the forefront of rapidly developing and exciting new chemistry.3-6 Notable milestones are the oxidative coupling of Ge94- clusters to oligomers and infinite chains,7-19 their metallation,14-16,20-25 capping by transition-metal organometallic fragments,26-34 insertion of a transition-metal atom at the center of the cluster which is sometimes combined with capping and oligomerization,35-47 addition of main-group organometallic fragments as exo-bonded substituents,48-50 and functionalization with various organic residues by reactions with organic halides and alkynes.51-58 
This latter development of attaching organic fragments directly to the clusters has opened up a new field, namely organo-Zintl chemistry, that is potentially fertile for further synthetic explorations, and it is the step-by-step procedure for the synthesis of germanium-divinyl clusters described herein. The initial steps outline the synthesis of an intermetallic precursor of K4Ge9 from which the Ge94- clusters are extracted later in solution. This involves fused-silica glass blowing, arc-welding of niobium containers, and handling of highly air-sensitive materials in a glove box. The air-sensitive K4Ge9 is then dissolved in ethylenediamine in the box and then alkenylated by a reaction with Me3SiC&equiv;CSiMe3. The reaction is followed by electrospray mass spectrometry while the resulting solution is used for obtaining single crystals containing the functionalized clusters [H2C=CH-Ge9-CH=CH2]2-. For this purpose the solution is centrifuged, filtered, and carefully layered with a toluene solution of 18-crown-6. Left undisturbed for a few days, the so-layered solutions produced orange crystalline blocks of [K(18-crown-6)]2[Ge9(HCCH2)2]&bull;en which were characterized by single-crystal X-ray diffraction. 
The process highlights standard reaction techniques, work-up, and analysis towards functionalized deltahedral Zintl clusters. It is hoped that it will help towards further development and understanding of these compounds in the community at large.

Watch this Scientific Journal Video about Synthesis of Nine-atom Deltahedral Zintl Ions of Germanium and their Functionalization with Organic Groups at JoVE.com

Synthesis of Nine-atom Deltahedral Zintl Ions of Germanium and their Functionalization with Organic Groups

We present the high-temperature synthesis of intermetallic precursors K4Ge9, their dissolution in ethylenediamine to form Ge94- deltahedral Zintl ions, and the reaction of the clusters with alkynes to form organo-Zintl ions. The latter are characterized by electrospray mass spectrometry in solutions and by single-crystal X-ray diffraction in the solid state.

Although the first studies of Zintl ions date between the late 1890's and early 1930's they were not structurally characterized until many years later.1,2 ...

synthesis-nine-atom-deltahedral-zintl-ions-germanium-their

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13.9K Views.  University of Notre Dame. We present the high-temperature synthesis of intermetallic precursors K4Ge9, their dissolution in ethylenediamine to form Ge94- deltahedral Zintl ions, and the reaction of the clusters with alkynes to form organo-Zintl ions. The latter are characterized by electrospray mass spectrometry in solutions and by single-crystal X-ray diffraction in the solid state.

Video: Synthesis of Nine-atom Deltahedral Zintl Ions of Germanium and their Functionalization with Organic Groups

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

High-Throughput Measurement and Classification of Organic P in Environmental Samples

Many types of organic phosphorus (P) molecules exist in environmental samples1. Traditional P measurements do not detect these organic P compounds since they do not react with colorimetric reagents2,3. Enzymatic hydrolysis (EH) is an emerging method for accurately characterizing organic P forms in environmental samples4,5. This method is only trumped in accuracy by Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy (31P-NMR) -a method that is expensive and requires specialized technical training6. We have adapted an enzymatic hydrolysis method capable of measuring three classes of phosphorus (monoester P, diester P and inorganic P) to a microplate reader system7. This method provides researchers with a fast, accurate, affordable and user-friendly means to measure P species in soils, sediments, manures and, if concentrated, aquatic samples. This is the only high-throughput method for measuring the forms and enzyme-lability of organic P that can be performed in a standard laboratory. The resulting data provides insight to scientists studying system nutrient content and eutrophication potential.

This protocol describes a high-throughput method of enzymatic hydrolysis that utilizes a microplate reader to measure and classify soil phosphorus as P monoesters, P diesters and inorganic P. Up to 96 samples can be measured at one time in a standard laboratory.

Many types of organic phosphorus (P) molecules exist in environmental samples1.  Traditional P measurements do not detect these organic P compounds since ...

FSL Constructs: A Simple Method for Modifying Cell/Virion Surfaces with a Range of Biological Markers Without Affecting their Viability

The ability to modify/visualize biological surfaces, and then study the modified cell/virion in a range of in vitro and in vivo environments is essential to gaining further insight into the function of specific molecules or the entire entity. Studies of biological surface modification are generally limited to genetic engineering of the organism or the covalent attachment of chemical moieties to the cell surface1,2. However these traditional techniques expose the cell to chemical reactants, or they require significant manipulation to achieve the desired outcome, making them cumbersome, and they may also inadvertently affect the viability/functionality of the modified cell. A simple method to harmlessly modify the surface of cells is required.
Recently a new technology, KODE Technology has introduced a range of novel constructs consisting of three components: a functional head group (F), a spacer (S) and a lipid tail (L) and are known as Function-Spacer-Lipid or FSL constructs3. The spacer (S) is selected to provide a construct that is dispersible in water, yet will spontaneously and stably incorporate into a membrane. 
FSL construct functional moieties (F) so far include a range of saccharides including blood group-related determinants, sialic acids, hyaluronan polysaccharides, fluorophores, biotin, radiolabels, and a range of peptides3-12. FSL constructs have been used in modifying embryos, spermatozoa, zebrafish, epithelial/endometrial cells, red blood cells, and virions to create quality controls systems and diagnostic panels, to modify cell adhesion/ interaction/ separation/ immobilization, and for in vitro and in vivo imaging of cells/virions3-12.
The process of modifying cells/virions is generic and extremely simple. The most common procedure is incubation of cells (in lipid free media) with a solution for FSL constructs for 1-2 hours at 37&deg;C4-10. During the incubation the FSL constructs spontaneously incorporate into the membrane, and the process is complete. Washing is optional. Cells modified by FSL constructs are known as kodecytes6-9, while virions are kodevirions10.
FSL constructs as direct infusions and kodecytes/kodevirions have been used in experimental animal models7,8,10. All kodecytes/kodevirions appear to retain their normal vitality and functionality while gaining the new function of the F moiety7,8,10,11.
The combination of dispersibility in biocompatible media, spontaneous incorporation into cell membranes, and apparent low toxicity, makes FSL constructs valuable research tools for the study of cells and virions.

Function-Spacer-Lipid (FSL) constructs allow the surface characteristics of living cells and virions to be modified without loss of vitality. The method requires only simple contact of an FSL construct solution with a cell/virion and spontaneous and stable surface incorporation occurs.

The ability to modify/visualize biological surfaces, and then study the modified cell/virion in a range of in vitro and in vivo environments is essential ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the structure of native control circuits. In both cases, the ability to program gene expression in a rapid and tunable fashion, with no off-target effects, can be useful. We have constructed yeast strains containing the ACT1 promoter upstream of a URA3 cassette followed by the ligand-binding domain of the human estrogen receptor and VP16. By transforming this strain with a linear PCR product containing a DNA binding domain and selecting against the presence of URA3, a constitutively expressed artificial transcription factor (ATF) can be generated by homologous recombination. ATFs engineered in this fashion can activate a unique target gene in the presence of inducer, thereby eliminating both the off-target activation and nonphysiological growth conditions found with commonly used conditional gene expression systems. A simple method for the rapid construction of GFP reporter plasmids that respond specifically to a native or artificial transcription factor of interest is also provided.

This protocol describes an experimental procedure for the rapid construction of artificial transcription factors (ATFs) with cognate GFP reporters&#160;and quantification of the ATFs ability to stimulate GFP expression via flow cytometry.

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase synthesis using standard phosphoramidite chemistry. Oligonucleotides are post-synthetically labeled by two new photostable cyanine dyes using copper-catalyzed click-chemistry. The synthesis of both donor and acceptor dye is described and is performed in three consecutive steps. With the DNA as the surrounding architecture, these two dyes undergo an energy transfer when they are brought into close proximity by hybridization. Therefore, annealing of two single stranded DNA strands is visualized by a change of fluorescence color. This color change is characterized by fluorescence spectroscopy but can also be directly observed by using a handheld ultraviolet (UV) lamp. The concept of a dual fluorescence color readout makes these oligonucleotide probes excellent tools for molecular imaging especially when the described photostable dyes are used. Thereby, photobleaching of the imaging probes is prevented, and biological processes can be observed in real time for a longer time period.

Photostable cyanine dyes are attached to oligonucleotides to monitor hybridization by energy transfer.